Comparative transcriptome analysis of five Medicago varieties reveals the genetic signals underlying freezing tolerance

Medicago plants such as M. sativa (lucerne, alfalfa) are important forage all over the world. Freezing-tolerance capacity is one of the key determinants of the survival and production of Medicago. In order to explore the molecular basis underlying freezing tolerance, we sequenced the root transcriptomes of five Medicago varieties belonging to two species, M. sativa and M. varia, and compared their gene expression and molecular evolution. A range of 19.5–23.8 Gb clean bases was obtained, and de novo transcriptome assembly generated 205 238–268 520 unigenes. The GO (Gene Ontology) terms of basic biological processes such as binding, cell and metabolism were most represented for the unigenes. In addition, a large number of unigenes related to GO terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of membrane, signalling, transcription and response to stimulus were identified in functional annotation. In total, 12 455 orthologs were identified among the five Medicago varieties. Among the orthologs, many unigenes that directly related to freezing tolerance were highly expressed in all five varieties, including genes for WRKY transcription factors, calcium-binding factors, and antioxidant enzymes such as catalase and ascorbate peroxidase. Molecular evolution testing showed that the unigenes involved in membrane shared high Ka/Ks (non-synonymous/synonymous substitution rate) across all the five Medicago varieties. Positively selected genes were mainly involved in transcription regulation, metabolism and signal transduction. Our study provides a large transcriptome dataset in the Medicago genus and brings new insights into the freezing tolerance for Medicago species.